During human trophoblast differentiation, mononuclear cytotrophoblast cells of the placental villous differentiate to form a multinucleated syncytium that synthesizes and secretes many growth factors and steroid hormones that are important maternal and fetal regulators. Our laboratory is studying the differentiation of primary cultures of villous cytotrophoblast cells, using the induction of human placental lactogen (hPL), human chorionic gonadotropin (hCG) and several other genes as fundamental markers of syncytiotrophoblast cell development. We have observed that there are two distinct phases of cytotrophoblast differentiation: morphological differentiation, during which the mononuclear cytotrophoblast cells fuse to form a syncytium, and biochemical differentiation, during which the syncytiotrophoblast cells develop differentiated functions such as hormone production. The overall hypothesis of this proposal is that morphological differentiation depends on transcription factors that induce syncytin, a protein known to be critical for syncytium formation in BeWo choriocarcinoma cells, and that biochemical differentiation depends on the activity of the transcription factor AP-2a, which we have shown to be critical for the induction of several syncytiotrophoblast genes. We postulate that the induction of AP-2a leads to transcriptional induction and repression of groups of downstream transcription factors and other target genes essential for syncytiotrophoblast function; and disruption of AP-2a action or syncytin expression may lead to placental disorders such as choriocarcinoma, hydatidiform mole and pre-eclampsia. The specific aims are to test the hypotheses that 1) the syncytin and AP-2cz genes are coordinately regulated during cytotrophoblast differentiation by cAMP, steroid hormones, and inflammatory cytokines; 2) the induction of syncytin expression is critical for syncytium formation during villous cytotrophoblast cell differentiation, and 3) AP- 2-dependent transcription factors and growth factors direct a genetic program that is critical for hormone production and other biochemical processes essential for human cytotrophoblast differentiation and function. Methodologies will include transfection studies, gel shift assays, and DNA microarray analyses. New knowledge about the genetic program that directs normal trophoblast differentiation may lead to the development of novel approaches for the treatment of placental tumors, pre-eclampsia, and other pathological conditions associated with abnormal placental development